Apoptosis is a morphologically distinct form of programmed cell death that plays important roles in development, tissue homeostasis and a wide variety of diseases, including cancer, AIDS, stroke, myopathies and various neurodegenerative disorders. Apoptosis occurs by activating an intrinsic cell suicide program which is constitutively expressed in most animal cells, and key components of this program have been conserved in evolution from worms to insects to man. A central step in the execution of apoptosis is the activation of a specific class of cysteine proteases, termed caspases that are widely expressed as inactive zymogens. The overall objective of the proposed research is to gain insight into the molecular mechanisms of caspase regulation and cell death, with particular emphasis on negative regulators of caspases. The specific focus of this proposal is on a set of ubiquitin pathway proteins that play a complex but specific role in regulating the onset of apoptosis via selective protein degradation. Inhibitor of Apoptosis Proteins (IAPs) are E3-ubiquitin ligases that protect cells against unwanted apoptosis by inhibiting caspases in live cells. However, upon death-inducing stimuli, IAPs auto-ubiquitinate and self-destruct in cells that are doomed to die. One major goal of this proposal is to define the precise mechanism by which the Drosophila pro-apoptotic Reaper protein inhibits IAPs. For this purpose, we will use a combination of site-directed mutagenesis, in vitro ubiquitination assays, protein interaction analyses, in vivo expression and cell biological studies. We will also investigate the mechanism by which mouse XIAP inhibits caspases, and study the role of this protein in apoptosis and tumor suppression in vivo by taking advantage of XIAP-mutant mice that we generated during the previous project period. Finally, we will investigate the mechanism by which a cullin-3-based E3 ubiquitin ligase complex that we recently identified from a genetic screen promotes caspase activation during sperm differentiation in Drosophila. This work should significantly advance our understanding of how caspases are regulated, and how their activity can be manipulated for therapeutic purposes. PUBLIC HEALTH RELEVANCE: This proposal focuses on negative regulation of caspases, the key executioners of apoptosis (programmed cell death), by a conserved family of ubiquitin pathway proteins. Progress during past grant periods has provided the intellectual basis for developing a novel class of small-molecule cancer therapeutics that are currently being evaluated in human clinical studies, and the results from proposed mouse model studies (aim 2) will have a direct influence on guiding these clinical trials. We will take a multi-disciplinary approach that integrates cellular, molecular genetic and biochemical studies in both Drosophila and the mouse. This is a uniquely powerful approach for taking a project from basic science discoveries to pre-clinical studies. At this time we are the only laboratory in the world that uses both Drosophila and mouse genetics to investigate the regulation of cell death, and to explore how abnormal regulation of this process contributes to human diseases, with a particular focus on cancer. Therefore, this project will have profound implications for both basic science and medicine.